Vaccinia virus is a prototypic member of the group known as poxviruses. Vaccinia virus is widely known and used as an expression vector of foreign gene or portions thereof. The majority of the foreign genes that have been inserted into the vaccinia virus genome encode antigens required for the production of immunity against a variety of infectious agents. It has been used to express a number of genes synthesizing proteins useful in the pharmaceutical/medical sector such as the hepatitis B surface antigen, the influenza virus hemagglutinin and the herpes virus glycoprotein D, neomycin-resistant gene and rabies virus glycoprotein for the purpose of foreign protein and vaccine production. After infection, immune response is elicited against both the vaccinia virus and the foreign protein.
The general procedure to produce recombinant virus expressing the foreign gene is to transfect target cells with a recombinant transfer vector containing the foreign gene of interest and with vaccinia virus. During the replication of the virus in the cell, homologous recombination takes place between some of the viral and the transfected DNAs resulting in a progeny virus containing foreign gene of interest as part of its genome. Classically, foreign DNA was inserted into a region of the vaccinia virus genome such as the endogenous thymidine kinase gene of wild-type virus. Panicali and Paoletti (Proc. Natl. Acad. Sci. U.S.A., 79, 4927-4931 1982) inserted the herpes virus thymidine kinase (TK) gene into a number of vaccinia virus preparations and obtained vaccinia recombinants expressing the herpes virus TK gene. The viral infectivity was shown not to be impaired by the presence of the foreign gene in sharp contrast to SV40 and retrovirus expression systems (Smith and Moss, Gene, 25, 21-28, 1983). These authors also suggested that vaccinia virus expression vector synthesizing proteins of pathogenic organisms might also be employed for medical and vaccination practices. The first candidate vaccine of this type expressed the hepatitis B surface antigen. Since then recombinant vaccinia virus has been employed in the vaccination of rodents against rabies. Piccini and Paoletti (Advances in Virus Research, 34, 43-64, 1989) have recently described the features and advantages of the vaccinia virus expression system and have outlined a number of proteins synthesized by the recombinant virus for the purpose of immunization.
Thus vaccinia virus has been successfully utilized as an expression vector through the insertion of foreign genes into a non-essential region of the viral genome via homologous recombination. However, one serious drawback afflicts the system; vaccinia virus expression vectors are not capable of producing abundant amounts of foreign proteins because of the lack of known strong promoter.
Entomopoxviruses are a group of poxviruses that have been found only in insect species. They posses many of the features of vertebrate poxviruses but are distinguished by the presence of large proteinic cytoplasmic occlusion bodies in which the virions are embedded at the end of the replication cycle (Arif, B.M., Advances in Virus Research, 29, 195-211, 1984). The occlusion bodies are composed mainly of a major vital-coded protein called spheroidin having a molecular weight of approximately 50,000 Daltons (Yuen, Dionne, Arif and Richardson, Virology, for occlusion of the virions and to afford the virions a certain amount of protection against inactivating environmental hazards such as ultraviolet light, desiccation, heat etc. Upon ingestion of the occlusion bodies by a susceptible larva, the gut juices, which are alkaline, dissolve the occlusion bodies thus releasing the virus to cause infection in the larva. Spheroidin is not essential for virus replication but because of its role in viral occlusion and protection, it is abundantly expressed late in the infection cycle. The spheroidin gene of the Choristoneura biennis entomopoxvirus has been isolated, cloned and sequenced (Yuen, Dionne, Arif and Richardson, Virology, 175, 427-433, 1990 ). The gene possesses a powerful promoter required for abundant synthesis of spheroidin. In fact, at the end of the replication cycle of the virus in the insect, approximately 20-50% of the larval protein is spheroidin. This promoter appears to be much more powerful than any of the vaccinia late genes promoters.
Thus the recombinant vertebrate poxvirus expression vectors constructed so far suffer from a drawback that these systems often lack a strong promoter and require very strict conditions in order to obtain adequate expression of the foreign gene. Therefore, engineering of a vertebrate poxvirus expression system powered by a promoter to substantially increase the level of expression has been long desired.